The English interrogative reverses the usual Subject-Verb order:
Now are you ready to play?
Now, are you ready to play?
Let's talk about a hypothetical: a coach talking to members of his team.
If you use a comma, the "now" is introductory, along the lines of "Now let's get to the important question." We can suppose that the coach is talking to a player who has been cutting practice and goofing off. He wants to know whether the player is going to start taking the team seriously.
If you don't use a comma, the "now" is a question about the immediate present, that is, right now. In this case, we can think of the coach finishing a pep talk to his team, who are behind at the half and just about to play the second half of the game. The coach wants to know if the team is ready for the immediately-upcoming challenge of the rest of the game.